Inhibition of proteolysis by the proform of eosinophil major basic protein (proMBP) requires covalent binding to its target proteinase.

By proteolytic cleavage of insulin-like growth factor binding proteins, the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) is able to control the biological activity of insulin-like growth factors. PAPP-A circulates in pregnancy as a proteolytically inactive complex, disulfide bound to the proform of eosinophil major basic protein (proMBP). We here demonstrate that co-transfection of mammalian cells with PAPP-A and proMBP cDNA results in the formation of a covalent PAPP-A/proMBP complex in which PAPP-A is inhibited. Formation of the complex also occurs when PAPP-A and proMBP synthesized separately are incubated. Complex formation was monitored by Western blotting, and by using an immunoassay specific for the complex. Using mutagenesis, we further demonstrate that the complex forms in a specific manner and depends on the presence of two proMBP cysteine residues. Mutated proMBP, in which Cys-51 and -169 are replaced by serine, is unable to form the covalent complex with PAPP-A. Of particular interest, such mutated proMBP further lacks the ability to inhibit PAPP-A. For the first time, this conclusively demonstrates that proMBP is a proteinase inhibitor. We further conclude that proMBP inhibits PAPP-A in an unusual manner, not paralleled by other proteinase inhibitors of our knowledge, which requires proMBP to be covalently bound to PAPP-A by disulfide bonds. ProMBP binding to PAPP-A most likely either abrogates substrate access to the active site of PAPP-A or induces a conformational change in the structure of PAPP-A, as we, by further mutagenesis, were able to exclude that the inhibitory mechanism of proMBP is based on a cysteine switch-like mechanism.

Mutations Phe785Leu and Thr618Met in Na+,K+-ATPase, associated with familial rapid-onset dystonia parkinsonism, interfere with Na+ interaction by distinct mechanisms.

The Na(+),K(+)-ATPase plays key roles in brain function. Recently, missense mutations in the Na(+),K(+)-ATPase were found associated with familial rapid-onset dystonia parkinsonism (FRDP). Here, we have characterized the functional consequences of FRDP mutations Phe785Leu and Thr618Met. Both mutations lead to functionally altered, but active, Na(+),K(+)-pumps, that display reduced apparent affinity for cytoplasmic Na(+), but the underlying mechanism differs between the mutants. In Phe785Leu, the interaction of the E(1) form with Na(+) is defective, and the E(1)-E(2) equilibrium is not displaced. In Thr618Met, the Na(+) affinity is reduced because of displacement of the conformational equilibrium in favor of the K(+)-occluded E(2)(K(2)) form. In both mutants, K(+) interaction at the external activating sites of the E(2)P phosphoenzyme is normal. The change of cellular Na(+) homeostasis is likely a major factor contributing to the development of FRDP in patients carrying the Phe785Leu or Thr618Met mutation. Phe785Leu moreover interferes with Na(+) interaction on the extracellular side and reduces the affinity for ouabain significantly. Analysis of two additional Phe(785) mutants, Phe785Leu/Leu786Phe and Phe785Tyr, demonstrated that the aromatic function of the side chain, as well as its exact position, is critical for Na(+) and ouabain binding. The effects of substituting Phe(785) could be explained by structural modeling, demonstrating that Phe(785) participates in a hydrophobic network between three transmembrane segments. Thr(618) is located in the cytoplasmic part of the molecule near the catalytic site, and the structural modeling indicates that the Thr618Met mutation interferes with the bonding pattern in the catalytic site in the E(1) form, thereby destabilizing E(1) relative to E(2)(K(2)).